Clinical Applications of Monoclonal

Clinical Applications of Monoclonal

Antibodies
Antibodies
(Mabs)
(Mabs)

Akanksha
AkankshaSingh
Singh
 Overview
Overviewofof
Presentation
Presentation
Introduction

History

Structure of Human and therapeutic Antibodies

Production of MAbs

Mabs Vs Polyclonal Abs

Application of Monoclonal Antibody

Market of Pharmaceutical Antibodies

EngineeredAntibodies
 Introduction
Introduction
Humans and all jawed vertebrates have the ability to make antibodies which are able to recognize (by
Humans
binding to) and all jawed
virtually vertebrates
any antigenic have the ability
determinant to make
(epitope) antibodies
and to which
discriminate are ableeven
between to recognize
similar (by
binding to) virtually any antigenic determinant (epitope) and to discriminate between even similar
epitopes.
epitopes.

Monoclonal antibodies (MAb or moAb) are monospecific antibodies that are the same because they

areMonoclonal
all clones ofantibodies (MAb or moAb) are monospecific antibodies that are the same because they
a B cell and have identical paratopes.
are all clones of a B cell and have identical paratopes.

High specificity in binding

High specificity in binding
Can activate and couple components of the immune
Can activate and couple components of the immune
system
Unique features of system
Unique features
Monoclonal of
Antibodies Can be used to treat serious diseases like multiple
Monoclonal Antibodies Can berheumatoid
sclerosis, used to treat seriousand
arthritis diseases liketypes
different multiple
of
sclerosis, rheumatoid arthritis and different types of
cancer
cancer

Production of monoclonal antibodies (MAbs) to an

Production
almost of monoclonal
endless antibodies
variety of antigenic (MAbs) to an
substances
almost endless variety of antigenic substances

Modification to structure and refinement in production methods have made antibodies a viable modern
Modification to structure and refinement in production methods have made antibodies a viable modern
drug.
drug.
 History
History

Discovery of
Immunization chemical
Emil von Behring structure of
developed antibody
serum therapy In 1961, Gerald
as an effective Edelman and
treatment Rodney Porter
against diptheria gave chemical
and tetanus structure of
antibody

In 1986, OKT- 3 was first monoclonal antibody to be approved for use in organ transplant rejection.
In 1986, OKT- 3 was first monoclonal antibody to be approved for use in organ transplant rejection.
 Structure of Human and therapeutic Antibodies
Structure of Human and therapeutic Antibodies

• Antibodies are a key component of the adaptive immune response, playing a central role

in both in the recognition of foreign antigens and the stimulation of an immune response
to them.
• 2 identical light chains (~220 amino acids long)
• Variable domain: VL
• Constant domain: CL

• 2 identical heavy chains (~440 amino acids long)

• Variable domain: VH
• 3 ConstantHeavy
domains: CH1, CH2, CH3
Heavy
Chains
Chains Antigen Constant
• Covalent, disulfide bonds between cysteine residues Constant
• Flexible “hinge region” Antigen
binding site region
binding site region

Hinge
Hinge
region
region

Light
Light
Chain
 AADynamic
DynamicBinding
BindingSite
Site

• The functional groups of the paratope (Fab) interact with the epitope
(antigen)
– Hydrogen bonding
– Van der Waals forces
– Ionic interactions
• The CDRs (Complementarity-Determining Region) are necessary for
antigen binding.
• The tertiary structure of this region can contain pockets, undulating flatter
surfaces, and even protrusions.
• Small antigens typically bind in deep pockets

• For some therapeutic mAbs, the affinity must be balanced so that effective
antigen binding occurs while tissue
penetration is allowed.
Production
ProductionofofMAbs
MAbs
Hybridoma Technology
Hybridoma Technology
Step 1: - Immunization Of Mice & Selection Of Mouse
Donor For Generation Of Hybridoma cells

ANTIGEN ( Intact cell/

Whole cell membrane/
micro-organisms ) +
ADJUVANT
(emulsification)

Ab titre reached in Serum

Spleen removed
(source of cells)
 PRODUCTION
PRODUCTIONOF
OFMONOCLONAL
MONOCLONALANTIBODY
ANTIBODY

Step 2: - Screening Of Mice For Antibody Production

After several
weeks of
immunization

Serum Antibody Titre Determined

(Technique: - ELISA / Flow
cytometery)

Titre too low Titre High

2 weeks
BOOST BOOST
(Pure antigen) (Pure antigen)
 PRODUCTION
PRODUCTIONOF
OFMONOCLONAL
MONOCLONALANTIBODY
ANTIBODY

Step 3: - Preparation of Myeloma Cells

Myeloma Cells
+ 8Azaguanine
-

Immortal Tumor Of Lymphocytes

Myeloma Cells
HGPRT-
High Viability & Rapid Growth
 PRODUCTION
PRODUCTIONOF
OFMONOCLONAL
MONOCLONALANTIBODY
ANTIBODY

Step 4: - Fusion of Myeloma Cells with Immune Spleen

Cells &
Selection of Hybridoma Cells
PEG
FUSION
SPLEEN CELLS MYELOMA CELLS
Feeder Cells
Growth Medium

1. Plating of Cells
in HAT selective
Medium
HYBRIDOMA CELLS
ELISA PLATE 2. Scanning of
HAT Medium Viable
Hybridomas
 PRODUCTION
PRODUCTIONOF
OFMONOCLONAL
MONOCLONALANTIBODY
ANTIBODY

Step 4: - Cloning of Hybridoma Cell Lines by “ Limiting

Dilution” or Expansion

A. Clone Each +ve

Culture
B. Test Each Supernatant for
Antibodies

C. Expand +ve
Clones

Mouse
Tissue
Ascites
Culture
Method
Method
PRODUCTION
PRODUCTIONOF
OFMONOCLONAL
MONOCLONALANTIBODY
ANTIBODY
 Monoclonal
MonoclonalVs
VsPolyclonal
Polyclonal
Antibodies
Antibodies
POLYCLONAL MONOCLONAL
POLYCLONAL MONOCLONAL
Derived from different B Derived from a single B
Derived from different B
Lymphocytes cell lines cell clone
Lymphocytes cell lines
mAb offer Reproducible,
Batch to Batch variation Predictable & Potentially
affecting Ab reactivity & inexhaustible supply of Ab
titre with exquisite specificity

NOT Powerful tools for Enable the development of

clinical diagnostic tests secure immunoassay
systems.
 Application
ApplicationofofMAbs
MAbs

Diagnostic Appliations
Biosensors and Microarrays
Therapeutic Applications
Transplant rejection – Muronomab – CD3
Cardiovascular disease - Abciximab
Cancer - Rituximab

Clinical Applications
Purification of the drug
Imaging the target
 Market
MarketofofPharmaceutical
Pharmaceutical
Antibodies
Antibodies

Fastest growing segment of Biopharmaceutical market with an

average annual growth of 18%.

Monoclonal Antibody market in 2010 was of $30 Billion and

Mabs, in vitro diagnostic market was that of $ 34 billion.

Currently there are approx 500 antibody based therapies under

development and more than 20 therapeutic Mabs on the market.
 Engineered
EngineeredAntibodies
Antibodies

• Small antibody fragments (Fv or Fab) are also

effective in blocking cytokines
– Benefit: More readily penetrate tissue
• Coupling of antibody fragments to form dimers and
tetramers
– Increases avidity and cross-linking

• Nanobodies
– 1989 - Raymond Hamers
– Discovered in camels
– Completely lack the light chain
– Same antigen affinity as their four-chain
counterparts
– Structure makes them more resistant to
heat and pH
• May lead to development of oral
nanobody pills
 References
References

Alberts,
Alberts,Bruce,
Bruce,etetal.
al.Molecular
Molecularbiology
biologyofofthe
thecell.
cell.New
New
York:
York:Garland
GarlandScience,
Science,2002.
2002.

Payne, W.J. et al. (1988).Clinical Laboratory Applications of

Payne, W.J. et al. (1988).Clinical Laboratory Applications of
Monoclonal Antibodies. Clinical Microbiology reviews. P 313--329
Monoclonal Antibodies. Clinical Microbiology reviews. P 313--329
Thank you